Lisofylline analogs and methods for use

ABSTRACT

The present invention provides analogs of a Lysofylline (LSF), and synthetic methods for the preparation of such analogs. The have the active side chain moiety (5-R-hydroxyhexyl) of LSF and can have greater potency and oral bioavailability than LSF.

RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.12/036,646 filed on Feb. 25, 2008, now U.S. Pat. No. 8,481,580 which isa Continuation filed under 35 U.S.C. §111 (a) of PCT/US2006/033777 filedon Aug. 29, 2006 and published in English as WO 2007/027719 A2 on Mar.8, 2007; which International Application claims priority fromprovisional patent application Ser. No. 60/712,114, filed Aug. 29, 2005,these applications and publications are incorporated herein byreference.

GOVERNMENT FUNDING

The invention described herein was made with government support underGrant Numbers DK 63521 and R21DK 063521 awarded by the NationalInstitute of Health. The United States Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

Type 1 diabetes is an autoimmune disorder which results from theimmune-mediated inflammatory destruction of insulin-producing β-cells inpancreatic islets. Although the specific pathogenic mechanisms in Type 1diabetes are not known, it is believed that activated T cells andmacrophages are required for the initiation. Once activated, macrophagessecrete several inflammatory cytokines, such as interleukin 1β(IL-1β),interleukin 12(IL-12) and tumor necrosis factor α (TNF-α), and triggerinterferon-γ (IFN-γ) production from activated T cells (see Z. D. Yang,M. Chen, R. Wu, M. McDuffie, J. L. Nadler, Diabetoiogia, 2002, 45,1307-131.4). These cytokines are reported to be cytotoxic to β cells andenhance Th1-mediated inflammatory responses, which are believed to beresponsible for the β cell destruction (see M. Chen, Z. D. Yang, R. Wu,J. L. Nadler, Endocrinology, 2002, 143(6), 2341-2348).

The anti-inflammatory compound Lisofylline (LSF;1-(5-R-hydroxyhexyl)-3,7-dimethylxanthine)) has been shown to be able toprotect β-cells from multiple inflammatory cytokine-mediated injuries byits ability to maintain insulin secretory capability and cell viability.

Agents such as Lisofylline may have clinical utility in preventingβ-cell damage during the development of Type 1 diabetes. This hypothesisis supported by the studies that showed Lisofylline could significantlyreduce spontaneous Type 1 diabetes development in the non-obese diabetic(NOD) mouse (see Yang). However, the disadvantages of Lisofylline maylimit its clinical development because it is not orally bioavailable andhas relatively weak potency. The structure of LSF is Formula I.

Currently, there is a need for novel, potent, and selective agents whichThere is a long felt need in the art for small molecules based on theLisofylline backbone which have enhanced potency, selectivity, and oralbioavailability. The present invention satisfies this need.

SUMMARY

The present invention provides analogs of a Lysofylline (LSF), andsynthetic methods for the preparation of such analogs. The analogs canhave greater potency and oral bioavailability than LSF. The analogs havethe active side chain moiety (5-R-hydroxyhexyl) of LSF. The inventionalso includes derivatives of LSF. LSF has formula I:

The disclosed analogs can be substituted with a variety ofnitrogen-contained heterocyclic compounds or on the hydroxyl group ofthe side chain. Accordingly, the invention provides 1-substituted5-hexanol compounds having additional substitution on the hydroxylgroup. The compounds have the formula II:

where R¹ is hydrogen or a group having the formula —C(═O)R³, wherein R³is lower alkyl. R² is selected from the group consisting of

and n is 1 or 2, or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides analogs havingformula III:

where each R⁴ is independently hydrogen or —(CH₂)_(m)—OR¹, where R¹ ishydrogen or a group having the formula —C(═O)R³, wherein R³ is loweralkyl and m is an integer from 2 to about 22, provided that at least oneR⁴ group is not hydrogen.

In another embodiment, the present invention provides analogs havingformula IV:

where each R⁵ is independently hydrogen or—(CH₂)_(i)—(CHOR¹)—(CH₂)_(j)—CH₃, where R¹ is hydrogen or a group havingthe formula —C(═O)R³, wherein R³ is lower alkyl, i is an integer from 1to about 20, and j is an integer from 0 to about 20, provided that thesum of i and j in each R⁵ group is from 2 to about 22 and at least oneR⁵ group is not hydrogen.

In another embodiment, the present invention provides analogs of LSFhaving the ability to protect cell viability, particularly the abilityto protect pancreatic β-cells. Thus, the disclosed analogs can allow thepancreas to maintain its insulin secretory capability.

In another embodiment, the present invention provides analogs of LSFwhich are effective in treating Type 1 diabetes. In another embodiment,the present invention provides analogs of LSF which can inhibit thedevelopment of Type 1 diabetes.

In another embodiment, the present invention provides analogs of LSFwhich are effective in treating Type 1 diabetes. In another embodiment,the present invention provides analogs of LSF which can lead to reversalof type I diabetes by allowing the body to regenerate beta cells.

In another aspect, the present invention also provides:

a pharmaceutical composition comprising a compound of formula II,formula III, formula IV, or pharmaceutically acceptable salts thereof,and a pharmaceutically acceptable carrier or excipient (the compositionpreferably comprises an effective amount of the compound or salt);

a method of treating or preventing Type 1 diabetes, comprisingadministering to a mammal (e.g., a human) in need of such treatment, acompound of formula II, formula III, formula IV, or pharmaceuticallyacceptable salts thereof;

a method for protecting cell viability, particularly the ability toprotect pancreatic β-cells comprising contacting (in vitro or in vivo)the cells with an effective protective amount of a compound of formulaII, formula III, formula IV, or a pharmaceutically acceptable saltthereof;

a compound of formula II, formula III, formula IV, or a pharmaceuticallyacceptable salt thereof for use in medical treatment (e.g., thetreatment of Type 1 diabetes); and

the use of a compound of formula II, formula III, formula IV, or apharmaceutically acceptable salt thereof to prepare a medicament fortreating Type 1 diabetes in a mammal (e.g., a human).

The disclosed analogs can be useful in the treatment of inflammatory andautoimmune diseases. Non-limiting examples of such diseases includeatherosclerosis, type 2 diabetes, disorders associated with visceralobesity such as non-alcoholic steatohepatitis (NASH), multiplesclerosis, inflammatory bowel disease, psoriasis, rheumatoid arthritis,Alzheimer's disease and the like.

The invention also provides methods for testing the activity of thedisclosed compounds. Methods not disclosed are known to those ofordinary skill in the art. One of ordinary skill will appreciate thatmany techniques are available to determine whether the compounds producethe desired result.

The invention also provides a kit for administering the disclosedcompounds.

The invention also provides novel intermediates and processes disclosedherein that are useful for preparing analogs of formula II, formula III,or formula IV, including the generic and specific intermediates as wellas the synthetic processes described in the Charts and Examples herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depict exemplary compounds of the invention.

FIG. 2 illustrates a scheme for preparing compound CPW11.

FIGS. 3A-3D illustrate schemes for preparing analogs having an R² groupbased on 2,3-dihydro-phthalazine-1,4-dione.

FIG. 4 illustrates schemes for preparing2,3-dihydro-pyridazino[4,5-d]pyridazine-1,4-dione and a related analog.

FIGS. 5A-5P illustrate the β-cell protective effect LSF and additionalanalogs of the invention for mouse-origin β-TC6 cells and mouse isletcells.

FIG. 6 illustrates the release of insulin from mouse islet cells.

FIGS. 7A-7D illustrate the release of insulin from mouse islet cells.FIG. 7A illustrates the basal-stimulated Insulin Release. FIG. 7Billustrates the glucose-stimulated Insulin Release. FIG. 7C illustratesthe ATP concentrations and FIG. 7D illustrates the β-cell viability.

FIGS. 8 a-8 c illustrate the ability of the disclosed analogs tostimulate insulin release from β-cells.

FIGS. 9A-9P illustrate the ability of LSF and additional analogs toeffect insulin release in mouse-origin β-TC6 cells.

FIG. 10 illustrates the ability of disclosed compounds (analogs) toreduce STAT4 phosphorylation in murine splenocytes (Immuno-dot-blot).

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described herein.

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

For purposes of the description of this invention, the articles “a” and“an” are used herein to refer to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease, condition, or disorder, or the frequency with which such asymptom is experienced by a subject, or both, are reduced.

As used herein, an “analog” of a chemical compound is a compound that,way of example, resembles another in structure but is not necessarily anisomer (e.g., 5-fluorouracil is an analog of thymine).

A “control” cell, tissue, sample, or subject is a cell, tissue, sample;or subject of the same type as a test cell, tissue, sample, or subject.The control may, for example, be examined at precisely or nearly thesame time the test cell, tissue, sample, or subject is examined. Thecontrol may also, for example, be examined at a time distant from thetime at which the test cell, tissue, sample, or subject is examined, andthe results of the examination of the control may be recorded so thatthe recorded results may be compared with results obtained byexamination of a test cell, tissue, sample, or subject. The control mayalso be obtained from another source or similar source other than thetest group or a test subject, where the test sample is obtained from asubject suspected of having a disease or disorder for which the test isbeing performed.

A “test” cell, tissue, sample, or subject is one being examined ortreated.

A “pathoindicative” cell, tissue, or sample is one which, when present,is an indication that the animal in which the cell, tissue, or sample islocated (or from which the tissue was obtained) is afflicted with adisease or disorder.

A tissue “normally comprises” a cell if one or more of the cell arepresent in the tissue in an animal not afflicted with a disease ordisorder.

The terms “compound” or “analog,” are used interchangeably. As usedherein, these terms refer to any type of substance or agent that iscommonly considered a drug, or a candidate for use as a drug,combinations, and mixtures of the above, as well as polypeptides andantibodies of the invention.

As used herein, a “derivative” of a compound refers to a chemicalcompound that may be produced from another compound of similar structurein one or more steps, as in replacement of a hydrogen atom by an alkyl,acyl, or amino group.

As used herein, an “effective amount” means an amount sufficient toproduce a selected effect.

The use of the Word “detect” and its grammatical variants is intended torefer to measurement of the species without quantification, whereas useof the word “determine” or “measure” with their grammatical variants aremeant to refer to measurement of the species with quantification. Theterms “detect” and “identify” are used interchangeably herein.

As used herein a “disease” is a state of health of an animal wherein theanimal cannot maintain homeostasis, and wherein if the disease is notameliorated then the animal's health continues to deteriorate.

In contrast, as used herein a “disorder” in an animal is a state ofhealth in which the animal is able to maintain homeostasis, but in whichthe animal's state of health is less favorable than it would be in theabsence of the disorder. Left untreated, a disorder does not necessarilycause a further decrease in the animal's state of health.

As used herein, a “functional” analog or molecule is a molecule in aform in which it exhibits a property or activity by which it ischaracterized.

As used herein, the term “inhibit,” refers to the ability of thedisclosed compounds to reduce or impede a described function.Preferably, inhibition is by at least 10%, more preferably by at least25%, even more preferably by at least 50%, and most preferably, thefunction is inhibited by at least 75%.

As used herein, “instructional material(s)” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the disclosed analogs in the kitfor effecting alleviation of the various diseases or disorders recitedherein. Optionally, or alternately, the instructional material maydescribe one or more methods of alleviating the diseases or disorders ina cell or a tissue of a mammal. The instructional material of the kitmay, for example, be affixed to a container which contains theidentified compound invention or be shipped together with a containerwhich contains the identified compound. Alternatively, the instructionalmaterial may be shipped separately from the container with the intentionthat the instructional material and the compound be used cooperativelyby the recipient.

As used herein, the term “purified” and similar terms relate to anenrichment of a molecule or compound relative to other componentsnormally associated with the molecule or compound in a nativeenvironment. The term “purified” does not necessarily indicate thatcomplete purity of the particular molecule has been achieved during theprocess. A “highly purified” compound as used herein refers to acompound that is greater than 90% pure.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans. Examples of these and other pharmaceuticallyacceptable carriers are described in Remington's Pharmaceutical Sciences(1991, Mack Publication Co., New Jersey).

A “sample,” as used herein, refers preferably to a biological samplefrom a subject, including, but not limited to, normal tissue samples,diseased tissue samples, biopsies, blood, saliva, feces, semen, tears,and urine. A sample can also be any other source of material obtainedfrom a subject which contains cells, tissues, or fluid of interest. Asample can also be obtained from cell or tissue culture.

The term “standard,” as used herein, refers to something used forcomparison. For example, a standard can be a known standard agent orcompound which is administered or added to a control sample and used forcomparing results when measuring said compound in a test sample.Standard can also refer to an “internal standard,” such as an agent orcompound which is added at known amounts to a sample and is useful indetermining such things as purification or recovery rates when a sampleis processed or subjected to purification or extraction proceduresbefore a marker of interest is measured.

A “subject” of analysis, diagnosis, or treatment is an animal. Suchanimals include mammals, preferably a human.

As used herein, the terms “treating or treatment” includes prophylaxisof the specific disorder or condition, or alleviation of the symptomsassociated with a specific disorder or condition and/or preventing oreliminating said symptoms.

As used herein, a “prophylactic” treatment is a treatment administeredto a subject who does not exhibit signs of a disease or exhibits onlyearly signs of the disease for the purpose of decreasing the risk ofdeveloping pathology associated with the disease.

As used herein, a “therapeutic” treatment is a treatment administered toa subject who exhibits signs of pathology for the purpose of diminishingor eliminating those signs.

As used herein, a “therapeutically effective amount” of a compound oranalog is an amount of compound which is sufficient to provide abeneficial effect to the subject to which the compound is administered.

As used herein, the term “halogen” or “halo” includes bromo, chloro,fluoro, and iodo. The term “haloalkyl” as used herein refers to an alkylradical bearing at least one halogen substituent, for example,chloromethyl, fluoroethyl or trifluoromethyl and the like.

The term “lower alkyl” refers to alkyl groups having from one (1) to (6)carbon atoms. Typically, C₁-C₆ alkyl groups include, but are not limitedto, methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl,tert-butyl, pentyl, hexyl, and the like.

It will be appreciated by those skilled in the art that the disclosedcompounds having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound, which possessthe useful properties described herein, it being well known in the arthow to prepare optically active forms (for example, by resolution of theracemic form by recrystallization techniques, by synthesis fromoptically-active starting materials, by chiral synthesis, or bychromatographic separation using a chiral stationary phase) and how todetermine cADPR agonist or antagonist activity using the standard testsdescribed herein, or using other similar tests which are well known inthe art.

The compounds of the present invention may exist in tautomeric forms andthe invention includes both mixtures and separate individual tautomers.For example the following structure:

is understood to represent a mixture of the structures:

The term “pharmaceutically-acceptable salt” refers to salts which retainthe biological effectiveness and properties of the compounds of thepresent invention and which are not biologically or otherwiseundesirable. In many cases, the compounds of the present invention arecapable of forming acid and/or base salts by virtue of the presence ofamino and/or carboxyl groups or groups similar thereto.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl) amines, tri(substituted alkenyl) amines,cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl amines,aryl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group. Examples of suitable amines include, by way of exampleonly, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl)amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol,tromethamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-alkylglucamines, theobromine, purines, piperazine, piperidine,morpholine, N-ethylpiperidine, and the like. It should also beunderstood that other carboxylic acid derivatives would be useful in thepractice of this invention, for example, carboxylic acid amides,including carboxamides, lower alkyl carboxamides, dialkyl carboxamides,and the like.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

In another embodiment, the present invention provides kits for use inadministering or using the disclosed compounds.

In cases where compounds are sufficiently basic or acidic to form acidor base salts, use of the compounds as salts may be appropriate.Examples of acceptable salts are organic acid addition salts formed withacids which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate.Suitable inorganic salts may also be formed, including hydrochloride,sulfate, nitrate, bicarbonate, and carbonate salts.

Exemplary values listed below for radicals, substituents, and ranges,are for illustration only; they do not exclude other defined values orother values within defined ranges for the radicals and substituents.

For example, (C₁-C₆)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl and the like.

The compounds of the present invention are generally named according tothe IUPAC or CAS nomenclature system. Abbreviations which are well knownto one of ordinary skill in the art may be used (e.g. “Ph” for phenyl,“Me” for methyl, “Et” for ethyl, “h” for hour or hours, “rt” for roomtemperature, and “rac” for racemic mixture).

The invention also provides compounds of formula II, formula III, orformula IV, for use in medical therapy.

A value for R¹ is hydrogen or C(═O)R³, wherein methyl, ethyl or propyl.

Another value for R³ is methyl, or ethyl.

Another value for R³ is methyl.

Another value for R¹ is hydrogen.

A value for R² is

In one aspect, the disclosed analogs have the formulas:

and pharmaceutically acceptable salts thereof.

More analogs can have having the formulas:

Processes for preparing analogs of formula II, formula III, or formulaIV, are provided as further embodiments and are illustrated by thefollowing procedures in which the meanings of the generic radicals areas given above unless otherwise qualified. A general scheme forpreparing the disclosed analogs having formula II is provided in Scheme1:

Scheme 2 (FIG. 2) provides a synthetic scheme route to prepare analog11. The preparation of additional analogs having formula III, or formulaIV, are illustrated in FIGS. 3A-3D and FIG. 4.

The compounds of formula I can be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, nnny be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions (suspensions) orsterile powders comprising the active ingredient which are adapted forthe extemporaneous preparation of sterile injectable or infusiblesolutions or dispersions, optionally encapsulated in liposomes. In allcases, the ultimate dosage form should be sterile, fluid and stableunder the conditions of manufacture and storage. This dispersion orsolution may be formulated according to the known art, and may comprise,in addition to the active ingredient, additional ingredients such as thedispersing agents, wetting agents, or suspending agents. The liquidcarrier or vehicle can be a solvent or liquid dispersion mediumcomprising, for example, water, ethanol, a polyol (for example,glycerol, propylene glycol, liquid polyethylene glycols, and the like),vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.Other acceptable diluents and solvents include, but are not limited to,Ringer's solution, isotonic sodium chloride solution, and fixed oilssuch as synthetic mono- or di-glycerides. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile solutions are prepared by incorporating the active compound inthe required amount in the appropriate solvent with various of the otheringredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile solutions, the preferred methods of preparation are vacuumdrying and the freeze drying techniques, which yield a powder of theactive ingredient plus any additional desired ingredient present in thepreviously sterile-filtered solutions. The sterile solutions or powderscan be combined with suitable carriers and administered via injection.In addition, the sterile solutions or powders can be combined withsuitable carriers and/or propellants and administered via inhalation.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

Generally, the concentration of the compound(s) of formula I in a liquidcomposition, such as a solution or suspension, will be from about 0.1-25wt %, preferably from about 0.5-10 wt %. The concentration in asemi-solid or solid composition such as a gel or a powder will be about0.1-5 wt %, preferably about 0.5-2.5 wt %.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected hut also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

In general, however, a suitable dose will be in the range of from about0.5 to about 20 mg/kg, e.g., from about 1 to about 18 mg/kg of bodyweight per day, such as 3 to about 16 mg per kilogram body weight of therecipient per day, preferably in the range of 6 to 14 mg/kg/day, mostpreferably in the range of 9 to 11 mg/kg/day.

The compound is conveniently administered in unit dosage form; forexample, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.5 to about75 μM, preferably, about 1 to 50 μM, most preferably, about 2 to about30 μM. This may be achieved, for example, by the intravenous injectionof a 0.05 to 5% solution of the active ingredient, optionally in saline,or orally administered as a bolus containing about 1-100 mg of theactive ingredient. Desirable blood levels may be maintained bycontinuous infusion to provide about 0.01-5.0 mg/kg/hr or byintermittent infusions containing about 0.4-15 mg/kg of the activeingredient(s).

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byadministration of a plurality of tablets.

Compounds which are identified using any of the methods described hereinmay be formulated and administered to a subject for treatment of any ofthe diseases and disorders described herein. However, the use of thedisclosed compounds should not be construed to include only the diseasesand disorder described herein. Preferably, the subject is a human.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts.

Modification of pharmaceutical compositions suitable for administrationto humans in order to render the compositions suitable foradministration to various animals is well understood, and the ordinarilyskilled veterinary pharmacologist can design and perform suchmodification with merely ordinary, if any, experimentation. Subjects towhich administration of the pharmaceutical compositions is contemplatedinclude, but are not limited to, humans and other primates, and mammals,including commercially relevant mammals such as cattle, pigs, horses,sheep, cats, and dogs.

A pharmaceutical composition may be prepared, packaged, or sold in bulk,as a single unit dose, or as a plurality of single unit doses. As usedherein, a “unit dose” is a discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient which would be administered to a subject or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition will vary, depending upon the identity, size, and conditionof the subject treated and further depending upon the route by which thecomposition is to be administered. By way of example, the compositionmay comprise between 0.1% and 100% (w/w) active ingredient.

In addition to the active ingredient, a pharmaceutical composition mayfurther comprise one or more additional pharmaceutically active agents.Particularly contemplated additional agents include anti-emetics andscavengers such as cyanide and cyanate scavengers.

Controlled- or sustained-release formulations of the pharmaceuticalcompositions herein may be made using conventional technology.

In some cases, the dosage forms to be used can be provided as slow orcontrolled-release of one or more active ingredients therein using, forexample, hydropropylmethyl cellulose, other polymer matrices, gels,permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, or microspheres or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known to those of ordinary skill in theart, including those described herein can be readily selected for usewith the pharmaceutical compositions. Thus, single unit dosage formssuitable for oral administration, such as tablets, capsules, gelcaps,and caplets that are adapted for controlled-release are encompassed bythe present invention.

Most controlled-release formulations are designed to initially releasean amount of drug that promptly produces the desired therapeutic effect,and gradually and continually release of other amounts of drug tomaintain this level of therapeutic effect over an extended period oftime. In order to maintain this constant level of drug in the body, thedrug must be released from the dosage form at a rate that will replacethe amount of drug being metabolized and excreted from the body.

Controlled-release of an active ingredient can be stimulated by variousinducers, for example pH, temperature, enzymes, water, or otherphysiological conditions or compounds.

Powdered and granular formulations of a pharmaceutical preparation maybe prepared using known methods. Such formulations may be administereddirectly to a subject, used, for example, to form tablets, to fillcapsules, or to prepare an aqueous or oily suspension or solution byaddition of an aqueous or oily vehicle thereto. Each of theseformulations may further comprise one or more of dispersing or wettingagent, a suspending agent, and a preservative. Additional excipients,such as fillers and sweetening, flavoring, or coloring agents, may alsobe included in these formulations.

A formulation of a pharmaceutical composition suitable for oraladministration may be prepared, packaged, or sold in the form of adiscrete solid dose unit including, but not limited to, a tablet, a hardor soft capsule, a cachet, a troche, or a lozenge, each containing apredetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, a paste, a gel, a toothpaste, a mouthwash, acoating, an oral rinse, or an emulsion. The terms oral rinse andmouthwash are used interchangeably herein.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in a freeflowing form such as a powder or granular preparation, optionally mixedwith one or more of a binder, a lubricant, an excipient, asurface-active agent, and a dispersing agent. Molded tablets may be madeby molding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface-active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide for pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition which are suitablefor oral administration may be prepared, packaged, and sold either inliquid form or in the form of a dry product intended for reconstitutionwith water or another suitable vehicle prior to use.

Injectable formulations may be prepared, packaged, or sold in unitdosage form, such as in ampules or in multi dose containers containing apreservative. Formulations far parenteral administration include, butare not limited to, suspensions, solutions, emulsions in oily or aqueousvehicles, pastes, and implantable sustained-release or biodegradableformulations. Such formulations may further comprise one or moreadditional ingredients including, but not limited to, suspending,stabilizing, or dispersing agents. In one embodiment of a formulationfor parenteral administration, the active ingredient is provided in dry(i.e., powder or granular) form for reconstitution with a suitablevehicle (e.g., sterile pyrogen free water) prior to parenteraladministration of the reconstituted composition.

A pharmaceutical composition may be prepared, packaged, or sold in aformulation suitable for buccal administration. Such formulations may,for example, be in the form of tablets or lozenges made usingconventional methods, and may, for example, 0.1 to 20% (w/w) activeingredient, the balance comprising an orally dissolvable or degradablecomposition and, optionally, one or more of the additional ingredientsdescribed herein. Alternately, formulations suitable for buccaladministration may comprise a powder or an aerosolized or atomizedsolution or suspension comprising the active ingredient. Such powdered,aerosolized, or aerosolized formulations, when dispersed, preferablyhave an average particle or droplet size in the range from about 0.1 toabout 200 nanometers, and may further comprise one or more of theadditional ingredients described herein.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. See Genaro, ed., 1985, Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., which is incorporated hereinby reference.

The compound can be administered to a subject as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, thetype, and age of the subject, etc.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thedisclosed pharmaceutical compositions. In accordance with oneembodiment, a kit is provided for treating a subject in need ofimmuno-modulation. Preferably, the subject is a human. In oneembodiment, the kit comprises one or more of the S1P analogs of thepresent invention and may also include one or more knownimmuno-suppressants. These pharmaceuticals can be packaged in a varietyof containers, e.g., vials, tubes, microtiter well plates, bottles, andthe like. Other reagents can be included in separate containers andprovided with the kit; e.g., positive control samples, negative controlsamples, buffers, cell culture media, etc. Preferably, the kits willalso include instructions for use.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are described herein.

In accordance with the present invention, as described above or asdiscussed in the Examples below, there can be employed conventionalclinical, chemical, cellular, histochemical, biochemical, molecularbiology, microbiology, and recombinant DNA techniques which are known tothose of ordinary skill in the art. Such techniques are fully explainedin the literature.

EXAMPLES

The invention is now described with reference to the following Examplesand Embodiments. Without further description, it is believed that one ofordinary skill in the art can, using the preceding description and thefollowing illustrative examples, make and utilize the present inventionand practice the claimed methods. The following working examplestherefore, are provided for the purpose of illustration only andspecifically point out some embodiments of the present invention, andare not to be construed as limiting in any way the remainder of thedisclosure. Therefore, the examples should be construed to encompass anyand all variations which become evident as a result of the teachingprovided herein.

Example 1 LSF Analogs

The side chain moiety (5-R-hydroxyhexyl) was kept constant and a varietyof nitrogen-contained heterocyclic compounds, including xanthine-like(5-aza-7-deazaxanthine-LSF) and non-xanthine-like skeletons, wereprepared for substitution with the hydroxyhexyl group. Most of theheterocyclic compounds were commercially available.5-aza-7-deazaxanthine in compounds 10 and 11), which was synthesizedfrom a 1,3,5-triazine derivative, cyanuric chloride. See J. D. Hepworth.Org. Synth. 5, 27-29; R. Calabretta, C. Giordano, C. Gallina, V. Morea,V. Consalvi and R. Scandurra. Eur. Med. Chem. 1995, 30, 931-941; S.Horrobin. J. Chem. Soc. 1963, 4130-4145; and S. H. Kim, D. G,Bartholomew, and L. B. Allen. J. Med. Chem. 1978, 21(9), 883-888. Thegeneral scheme for synthesis of the LSF analogs is provided in scheme 1.Exemplary compounds are depicted in FIG. 1. The synthesis of compound 11(CPW11) is illustrated in Scheme 2, FIG. 2.

Example 2 Biological Evaluations of LSF Analogs

The disclosed analogs (CPW1-15) were evaluated in pancreatic β celllines for apoptosis protection after treatment with inflammatorycytokines (reflected by reduced OD450) and insulin-release (see FIGS. 5and 6). For the protective effect of β-cells, some analogs showedcomparative potency with LSF and additional analogs were able to protectcells at low concentrations (nm and lower; see FIG. 5). For the insulinrelease assay, some compounds demonstrated effects similar to those ofLSF in response to glucose (CPW1-15; FIG. 6).

Further evaluations were performed for some of the LSF analogs in MDSassays, solubility, oral bioavailability, and stability (see Table 1).

TABLE 1 rCYP450 Inhibition and Metabolic Stabilities Metabolic StabilityCompound Formula rCYP % Inhibition Percent No. Weight 1A2 2C9 2C19 2D63A4 Remaining CPW1 338.24 58% 86% 99% 80% 57% 0% CPW4 296.2 56% 38% 94%50% 32% 0% CPW6 342.47 13% 3% 14% 16% 14% 77% CPW7 271.29 12% 5% 12% 8%12% na CPW8 319.36 7% 5% 24% 10% 12% 45% CPW9 277.32 5% −12% 1% 2% −4%106% CPW11 280.32 13% 12% 21% 3% 0% 82% CPW12 262.3 18% 30% 30% 12% 4%75% CPW20 283.36 95% 86% 98% 73% 50% 19% CPW13 362.46 25% 92% 89% −28%−159% 38%

Analogs having greater than 50% inhibitory activity were considered tohave high activity; analogs having from 20-49% inhibitory activity wereconsidered to have medium activity; and analogs having less than 20inhibitory activity were considered to have low activity

Example 3 Biological Evaluations of LSF Analogs in Human Islets

Four compounds (CPW7, CPW8, CPW11, and CPW12) were also evaluated onhuman islets for the induction of insulin secretion, intracellular ATPconcentrations and the effect of reducing cell death exposed tocytokines (combination of human IL-1β, IFN-γ, and TNF-α). The resultsare illustrated FIGS. 7A-7D.

The results of the testing indicate that CPW12 indicates that there werepositive effects in β-cell protection and insulin release on mouse isletcell line, the analog shows low cytochrome p450 inhibition (it was asafe compound with low potential for drug interactions). The Caco-2permeability test results shows CPW12 could be orally absorbed from theGI track, which is consistent with good oral bioavailability. The CPW12analog shows acceptable stability and solubility.

In the evaluation on human islet, CPW12 shows positive effects toimprove insulin secretion in the presence of inflammatory cytokines andhas a beneficial effect in maintaining mitochondrial function and cellviability in human β-cells.

Example 4 Effects of LSF and Analogs on β-Cells

The effects of LSF analogs on β-cells in the mouse insulin-secretingINS-1 cell line were investigated. Cells were maintained in RPMI 1640medium (Life Technologies, Inc., Gaithersburg, Md.) supplemented with10% heat-inactivated FBS, 10 mm HEPES, 200 μm L-glutamine, 1 mm sodiumpyruvate, 5 nm 2-mercaptoethanol, 50 U/ml penicillin, and 50 μg/mlstreptomycin at pH 7.4. The cells were cultured at 37° C. in ahumidified incubator supplied with 5% carbon dioxide. Fresh medium wasreplaced every 2 days. The cells were plated at a density of 105/cm².Culture vessels were coated with poly-D-lysine and gelatin (Sigma, St.Louis, Mo.) to retain detached and dead cells so that seeding cellnumbers reflect the actual cell numbers after all treatment conditions.INS-1 cells were treated with the combination of recombinant mouse IL-1β(5 ng/ml), IFNγ (100 ng/ml), and TNFα (10 ng/ml; R&D Systems, Inc.,Minneapolis, Minn.) suspended in complete RPMI medium. LSF (provided byCell Therapeutics, Inc., Seattle, Wash.) and analogs were addedsimultaneously with the cytokines in complete RPMI medium. Alltreatments were performed for 18 hours.

The results (illustrated in FIGS. 8 a-8 c) showed that the LSF analogscould protect β-cells from cytokine-induced cell death. INS-1 cells weretreated with a combination of recombinant IL-1β, INFγ, and TNFα with10⁻⁵-10³ μM LSF and its analogs. At a concentration of 10⁻² μM, LSFshowed a maximum protective effect (FIG. 8 a). There was no furtherincrease in β-cell protection when the LSF concentration was increasedfrom 10⁻² to 10² μM. The LSF analogs showed some protective effects onβ-cells, especially compounds CPW11 (FIG. 8 b) and CPW12 (FIG. 8 c),which proved effective at low concentrations. Both of these compoundsshowed comparative activity with LSF at concentration of 10⁻² μM. Nofurther increases in β-cell protection were observed when analogconcentrations were increased.

Example 5 Effects of LSF and Analogs on Insulin Secretion

The effects of LSF analogs on insulin secretion in INS-1 cells with andwithout inflammatory cytokines. β-Cells were treated with apoptosisdetecting dye for 2-3 hours at room temperature. Apoptotic cells wererecognized with purple-red color under a microscope. After washing toeliminate free dye and adding dye release reagent, color density wasquantified by reading at OD 450 nM. At the end of treatment, cells werewashed with Krebs-Ringer-bicarbonate-HEPES buffer (KRB) containing 134mm NaCl, 4.7 mm KCl, 1.2 mm KH₂PO₄, 1.2 mm MgSO₄, 1.0 mm CaCl₂, 10 mmHEPES, and 0.1% BSA at 37° C., pH 7.4. The cells were preincubated inthe same buffer for 30 min, followed by 60-min incubation in KRBsupplemented with 15 mM D-glucose (J. T. Baker, Phillipsburg, N.J.). Thesupernatant was harvested and subjected to centrifugation to eliminateresidue cells. Insulin secreted into the supernatant was measured by RIAwith mouse insulin as a standard. The cells are maintained in basal (3mM) and glucose-stimulated (28 mM). Insulin release was observed by 20μM of LSF and its analogs. Insulin secretion in INS-1 cells: (a) LSF,(b) compound CPW11, and (c) compound CPW12. (+CG3=w/compound, w/ocytokines, 3 mM glucose; −C−G3=w/o compound, w/o cytokines, 3 mMglucose; +C−G28=w/compound, w/o cytokine, 28 mM glucose;−C−G28=w/compound, w/o cytokine, 28 mM glucose; +C+G3=w/compound,w/cytokines, 3 mM glucose; −C+G3=w/o compound, w/cytokines, 3 mMglucose; +C+G28=w/compound, w/cytokines, 28 mM glucose; −C+G28=w/ocompound, w/cytokines, 28 mM glucose). The results are illustrated inFIGS. 9 a-9 c.

Example 6 Development of LSF Analogs Based on CPW12

Schemes are provided for additional analogs of CPW12, (see FIGS. 3A-3D,Schemes 3A-3D and FIG. 4, Scheme 4). In FIGS. 3A-3D, Schemes 3A-3Dmodifications of the side chain moiety are illustrated. (See V. Cere, C.Mazzini, C. Paolucci, S. Pollicino and A. Fava J. Org. Chem., 1993, 58,4567-4571; C. Paolucci, S. Pollicino and A. Fava J. Org. Chem., 1995,60, 169-175; F. Sato, Y. Tomuro, H. Ishikawa and M. Sato. Chem. Lett.1980, 99-102; and G. Cerichelli, C. Grande, L. Luchetti and G. ManciniJ. Org. Chem., 1991, 56, 3025-3030.)

In FIG. 4, Scheme 4 a modification of the nuclear moiety (R²) isillustrated. (See N. Haider, E. Mavrokordatou and A. Steinwender. Syn.Comm. 1999, 29(9), 1577-1584.)

Example 7 Mouse-Insulin Secreting Cell Line β-TC6

Cell Preparation

The β-TC6 cell line is maintained in RPMI 1640 medium (LifeTechnologies, Rockville, Md.) supplemented with 10% heat inactivatedfetal bovine serum, 10 mM HEPES, 200 pM L-glutamine, 50 units/mlpenicillin and 50 μg/ml streptomycin at pH 7.4. The cells are culturedin a 37° C., humidified incubator supplied with 5% carbon dioxide. Freshmedia is replaced every two days. Unless otherwise stated, the cells areplated at a density of 10⁵/cm². Culture vessels (dishes and chamberslides) used for experiments were coated with poly-D-lysine and gelatin(Sigma, St. Louis, Mo.) to retain detached and dead cells so thatseeding cell numbers reflect the actual cell numbers after all treatmentconditions.

Cytokine and LSF Analog Treatment of B-TC6 Cells.

β-TC6 cells are treated with vehicle alone or with the combination ofrecombinant mouse IL-1β (5 ng/ml), IFN-γ (100 ng/ml) and TNF-α (10ng/ml) (R&D Systems, Minneapolis, Minn.) suspended in complete RPMImedium. LSF (Cell Therapeutics, Inc. Seattle, Wash.) or the analogs areadded simultaneously with the cytokines in complete RPMI medium in theconcentrations ranging 1 nM-20 pM. All treatments are conducted for 18hours. The results are illustrated in FIGS. 5A-8P.

Example 8 Static Insulin Secretion

At the end of treatment, cells are washed withKrebs-Ringer-bicarbonate-HEPES buffer (KR13) containing in mM: 134 NaCl,4.7 KCl, 1.2 KH₂P0₄, 1.2 MgS0₄, 1.0 CaCI₂, 10 HEPES, and 0.1% bovineserum albumin at 37° C., at a pH of 7.4. The cells are pre incubated inthe same buffer for 30 min followed by 60-min incubation in KRBsupplemented with 15 mM D-glucose (J. T. Baker, Phillisburg, N.J.). Thesupernatant is harvested and subjected to centrifugation to eliminateresidue cells. Insulin secreted into the supernatant is measured usingEIA with mouse insulin as a standard. The results are illustrated inFIGS. 9A-9P.

Example 9 STAT 4 Phosphorylation

Murine splenocytes are equally plated in 96-well plates and are treatedwith or without LPS (1.0 ng/ml) supplemented with LSF or a series ofanalogs for 18 hours. The protein lysates are transferred onto theHybond-P membranes, and are subsequently probed with a polyclonalantibody against phosphorylated STAT4. The hybridized membranes aresubjected to ECL and autoradiography. Samples are repeated intriplicate. The results are illustrated in FIG. 10.

Other methods which were used but not described herein are well knownand within the competence of one of ordinary skill in the art ofclinical, chemical, cellular, histochemical, biochemical, molecularbiology, microbiology and recombinant DNA techniques.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentinvention may be embodied in other specific forms without departing fromthe spirit or essential attributes thereof.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. All publications, patents, and patentdocuments cited in the specification are incorporated by referenceherein, as though individually incorporated by reference. In the case ofany inconsistencies, the present disclosure, including any definitionstherein will prevail. The invention has been described with reference tovarious specific and preferred embodiments and techniques. However, itshould be understood that many variations and modifications may be madewhile remaining within the spirit and scope of the invention.

What is claimed is:
 1. A compound having formula II:

wherein R¹ is hydrogen or a group having the formula —C(═O)R³, whereinR³ is hydrogen or lower alkyl, and R² is

and n is 1 or 2; or a pharmaceutically acceptable salt thereof.
 2. Thecompound of claim 1, wherein each R³ independently is hydrogen, methyl,ethyl or propyl.
 3. The compound of claim 2, wherein each R³independently is hydrogen, methyl, or ethyl.
 4. The compound of claim 3,wherein each R³ independently is hydrogen or methyl.
 5. The compound ofclaim 4, wherein each R³ is hydrogen.
 6. The compound of claim 1, havingthe formula:

and pharmaceutically acceptable salts thereof.
 7. A pharmaceuticalcomposition comprising a compound of claim 1, in combination with apharmaceutically acceptable carrier.
 8. A method of treating a diseaseor disorder in a mammal comprising the step of administering to themammal an effective amount of a compound of claim 1, or apharmaceutically acceptable salt thereof, wherein said disease ordisorder is selected from the group consisting of atherosclerosis, type1 diabetes, type 2 diabetes, disorders associated with visceral obesity,multiple sclerosis, inflammatory bowel disease, psoriasis, rheumatoidarthritis, and Alzheimer's disease, or any combination thereof.
 9. Themethod of claim 8, wherein the disease or disorder is selected from Type1 diabetes.
 10. The method of claim 8, wherein the Type 1 diabetes istreated by inhibiting the apoptosis of pancreatic β-cells in a mammal.11. The method of claim 10, wherein the apoptosis of β-cells is inducedby IL-1β, IFN-γ, and TNF-α, or any combination thereof.
 12. The methodof claim 11, wherein the IL-1β, IFN-γ, and TNF-α, or any combinationthereof are produced by Th1 T cells.